The aims of the research proposed are to better understand the molecular mechanisms of three enzymes involved in key reactions in carbohydrate metabolism. These reactions are important for the utilization of carbohydrates for metabolic energy and for carbohydrate storage. The enzymes under investigation are phosphoenolpyruvate carboxykinase (PEPCK), enolase and pyruvate kinase (PK). Recent results with these enzymes give better paradigms to elucidate and relate the structures with their respective activities and functions. Malfunctioning of these enzymes has been postulated to be causes for sudden infant death syndrome, diabetic clinical secondary abnormalities, muscular malfunctioning, and non-spherolytic hemolytic anemia. One such anemia is caused by a point mutation in pyruvate kinase. Two enzymes, PEPCK and PK, are metabolically controlled. Each of the enzymes utilizes the "high energy" metabolite phosphoenol-pyruvate (PEP) as substrate and they each have multimetal sites and putative multi-cation functions for each enzyme. A long term goal is to locate the sites for each of the cations and to describe the specific functions for each. A postulate is that they may serve as a means of control of catalytic activity. An important tool in elaborating the cation site and function for PEPCK appears to have been developed by placing the exchange-inert Co(III) at the catalytic site. Kinetic, thermodynamic and structural studies of this enzyme are being performed. Additional amino acids at the catalytic site are being sought and a system to perform site-directed mutagenesis experiments is being developed. The advent of the crystal structure of enolase has given us a new dimension in our studies. Many previous notions have proven correct but new ideas have developed and require challenge. The function of the cation at site II appears to be an inactive one. It is remote from the catalytic site, not partaking in ligand binding nor catalytic chemistry. Several active site amino acids have been located. An experimental system to perform site-directed mutagenesis to unravel the roles of specific amino acid residues and to address the mechanistic process of this enzyme is developed. Muscle PK is serving as a simple model for the allosteric yeast PK. Site- directed modifications of this enzyme will help to describe specific roles of amino acids in catalysis and regulation. Kinetic and binding studies with yeast PK are giving important information enabling the description of a specific coupled symmetry model to describe the allosteric behavior of this enzyme. Specific structural information at the catalytic site is being scrutinized. This enzyme is beginning to serve as a specific model to describe allostery in a comprehensive and descriptive manner. The work with these enzymes is aiding in the development of new experimental tools to understand the functions of some cations in enzymes, enzyme structure and function and detailed concepts of allosteric regulation. This information is important in the ability to modulate catalytic activity both in vivo and in vitro.